So-called antibody drugs using monoclonal antibodies for therapy have annual sales of beyond $30 billion and belong to the largest segment in the biotechnology-based pharmaceutical products as well as the most rapidly growing segment in the entire pharmaceutical industry. Up to the present, 23 types of full-size monoclonal antibodies and three types of monoclonal antibody fragments have been launched. Some of them have already become blockbusters having annual sales of beyond $1 billion. The number of monoclonal antibodies as drug candidates whose clinical trials have been initiated during the period from 1995 to 2007 has increased three times or more and still further increased (Non Patent Literature 1).
With a growth and development of such an antibody drug market, research and development have been accelerated with a view to designing and improving a molecule having a binding property to an antibody, immunoglobulin G or a protein containing an Fc region of immunoglobulin G (hereinafter, also referred to as antibodies and others). This is because such a molecule is useful for research and production of antibodies and others, in particular, expected to have a great demand in affinity chromatography used in a recovery/purification step of a process for producing antibody drugs. This is also because protein A, which is derived from Staphylococcus and frequently used presently for recovering and purifying antibodies and others, is recognized as being insufficient in view of stability and production cost. At present, various research and development approaches are made for obtaining molecules capable of binding to antibodies and others (Non Patent Literature 2). One of them is development of antibody-binding peptides. Some of examples are as follows.
Suzuki et al., identified a plurality of polypeptides exhibiting binding activity to an Fc region of human IgG by using a phage library displaying 7-residue or 12-residue linear peptides on filamentous bacteriophage M13 and the presence or absence of a binding property to Fc region of human IgG was determined by Enzyme Linked Immuno-Sorbent Assay (ELISA) (Patent Literature 1). They extracted a common sequence from the sequences identified and a peptide was prepared from the common sequence. The binding activity of the peptide not only to human IgG but also to an Fc region of IgG derived from a horse, sheep, rabbit, guinea pig, goat, cat, dog, cow, pig and mouse, was checked by ELISA.
DeLano et al., obtained a plurality of 20-residue cyclic peptides, which bind to human IgG competitively with protein A derived from yellow Staphylococcus, by using a phage library displaying a cyclic peptide, which is cyclized via a disulfide bond and represented by Xaai Cys Xaaj Cys Xaak (where i, j and k are integers satisfying the expression: i+j+k=18), on filamentous bacteriophage M13. They further extracted a common sequence from these peptides and cyclic peptide Fc-III of 13 residues (Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr) (SEQ ID NO: 165) was prepared. They found that cyclic peptide Fc-III shows a competitive inhibitory capacity of Ki=100 nM in the competitive reaction with protein A. They disclosed that the in-vivo Fab half-life period can be improved by combining a Fab fragment (which is an antigen binding site of IgG) with Fc-III to make a fusion protein, in an experiment using rabbits (Patent Literature 2 and Non Patent Literature 3). Dias et al., further prepared FcBP-2 by further introducing cyclization into cyclic peptide Fc-III by use of D-form and L-form Pro residue and succeeded in enhancing its binding property (Fc-III equilibrium dissociation constant, KD=185 nM) to IgG up to KD=2 nM (Non Patent Literature 4).
Fassina et al., screened a library of a synthesized tetrapolypeptide represented by (Arg Thr Xaa)4 Lys2 Lys Gly (SEQ ID NO: 166) having a branched structure ascribed to a Lys residue and prepared peptide TG19318, which competitively works with protein A (Non Patent Literature 5). They showed that TG19318 has a binding property of KD=300 nM to rabbit IgG, and further that IgG contained in the sera of human, bovine, horse, pig, mouse, rat, goat and sheep can be purified by affinity chromatography prepared by immobilizing TG19318 (Non Patent Literature 6).
Ehrlich et al., isolated a peptide exhibiting a binding property to a pFc′ fragment, which is obtained by digesting humanized IgG with pepsin, by using a phage library displaying 7-residue or 12-residue linear peptide on filamentous bacteriophage M13, in the same manner as in Suzuki et al. (Non Patent Literature 7).
Krook et al., prepared a peptide exhibiting a binding property to an Fc region of human IgG by using a phage library displaying 10 residue-long linear peptides on filamentous bacteriophage M13. They confirmed by ELISA that this peptide exhibits a strong binding property to IgG molecules derived from human and pig (Non Patent Literature 8).
Verdoliva et al., screened a library of a synthetic peptide represented by (Cys Xaa3)2 Lys Gly (SEQ ID NO: 167), into which a branched structure ascribed to a Lys residue and cyclization ascribed to a Cys residue are introduced, for a mouse monoclonal IgG, and prepared peptide FcRM exhibiting a binding property to a site near a hinge region. They further constructed affinity chromatography using immobilized FcRM, and reported on purification of IgG molecules derived from a mouse and a human (Non Patent Literature 9).
Sakamoto et al., prepared a peptide exhibiting a binding property to an Fc region of human IgG by using a phage library displaying a cyclic peptide represented by Cys Xaa7-10 Cys on T7 bacteriophage (Non Patent Literature 10). The peptide they prepared is different from the aforementioned IgG-binding peptides so far prepared since the peptide recognizes not a natural structure of an Fc region but a non-native structure of the Fc region produced by an acid treatment. Ito et al., disclosed that if this peptide is used, the content of a non-native structure produced by an acid treatment and contained in human antibody drugs, immunoglobulin preparations and IgG reagents can be checked (Patent Literature 3).
As described in the above, a plurality of antibody-binding peptides have been developed; however, molecular diversity of them may not be sufficient. This is because antibodies are used in a wide variety of needs in various industrial fields besides the aforementioned therapeutic applications, and molecular properties required for an antibody-binding molecule are not the same. In detecting, purifying, immobilizing or removing antibodies and others, antibody-binding molecules having properties suitable for individual situations must be used. To describe more specifically, antibody-binding molecules having appropriate properties are required in consideration of the sites of the antibodies and others to which an antibody-binding molecule binds, the specificity of an antibody-binding molecule to the binding site, the binding affinity of an antibody-binding molecule, association/dissociation control by changing conditions of e.g., a solution, whether or not an antibody-binding molecule binds to antibodies of a plurality of animal species or a specific animal species alone, properties such as solubility and stability and possibility of a large-scale production. In the case of a peptide, for example, whether a peptide contains a non-native amino acid residue or is constituted only of natural amino acid residues, whether a peptide has a linear, cyclic or branched chemical structure, whether a peptide forms a stable three-dimensional structure in a solution and whether a peptide can be used under a reductive environment are the items to be considered when adaptability of the peptide is determined.
Usually, the function of a short-chain polypeptide is enhanced by stabilization through cyclization via e.g., an intramolecular disulfide crosslinkage; however, a complicated chemical reaction is required for cyclization. In addition, a short-chain polypeptide does not exhibit a binding function, for example, under a reductive condition where a disulfide crosslinkage is not easily formed. As the reductive condition where a disulfide crosslinkage is not easily formed, for example, (1) cytoplasmic environment and (2) environment where a reducing agent is added in order to convert the thiol group of a cysteine residue of a target IgG or an Fc region to a free radical for e.g., chemical modification, are conceived.
With the growth of the antibody drug market, it has recently been strongly desired to further improve separation/purification techniques and analysis techniques of antibody molecules.
As to the analysis techniques, it has been desired to develop the following three techniques: (1) analysis technique for heterogeneity of a molecule caused by post-translational modification including sugar chain addition; (2) analysis technique for heterogeneity of an antibody molecule caused by conformational change; and (3) analysis technique for heterogeneity of a molecule caused by formation of associates or aggregates (Non Patent Literature 11). It has been reported that when an antibody molecule receives various physical or chemical stresses, a non-native structure (different from a general natural structure) called an alternatively folded state (AFS) is formed. It is suggested that such a non-native structure not only reduces the effect of a drug but also induces immunogenicity, causing a risk of a side effect. For the reason, an analysis technique for finding an antibody having a non-native structure is desired (Non Patent Literature 12).
Examples of a technique for analyzing the shape or conformation of a protein molecule include X-ray crystal structural analysis, nuclear magnetic resonance, electron microscopic analysis, analytical ultracentrifugation, isoelectric point electrophoresis, dynamic light scattering, circular dichroism spectroscopy and liquid chromatography; these methods all fail to satisfy both atomic-level accuracy and throughput. To describe more specifically, for example, the X-ray crystal structural analysis and nuclear magnetic resonance can provide conformational data with an atomic-level accuracy; however, several months are required for analysis. In contrast, in the dynamic light scattering and liquid chromatography, measurement is completed in several minutes; however, a small molecular change and slight contamination cannot be detected. In the circumstances, it has been desired to develop a simple technique satisfying not only an atomic-level accuracy but also throughput.
In the meantime, as the separation/purification technique, an affinity chromatography technique using a molecule having a specific affinity for an antibody as a ligand is presently indispensable (Non Patent Literature 13). At present, the ligand for use in affinity chromatography, a bacteria-derived natural protein such as protein A and protein G are used. These proteins have satisfactory affinity for antibodies; however, low stability and high production cost are drawbacks of them.